Ti liu 1. Sn xut ethanol sinh hc t li bp s dng Aspergillus niger v saccharomyces cerevisae bng phng php kt hp ng ha v ln men ng thi 2.

Cc ch phm enzyme thy phn li bp v sn xut ethanol t cellulose thy phn 3. Sn xut ethanol bng cch thy phn li bp nh Escherichia coli KO11 4. So snh s khc nhau gia hai phng php thy phn li bp sn xut ethanol bng Pichia stipitis vi Candida shehatae. 5. Enzyme lignase t Penicillium decumbens JU-A10???? 6. Thu nhn cellulase t Trichoderma viride c cht l cht thi nng nghip 7. Nng cao hiu sut enzyme chuyn ha v sn xut ng bng cch thy phn enzyme hai bc li bp t ph thi sn xut XYLO-oligosaccharides. 8. Tin x l b ma, li ng v rm r bng acid long sn xut ethanol bng saccharomyces cerevisiae 9. Sn xut cellulase t Trichoderma reesei vi c cht l mn ca 10. Sn xut cellulase t Aspergillus flavus Linn Isolate NSPR 101 bng cch ln men b ma, mn ca, li ng 11. Sn xut cellulase cht lng cao t Trichoderma reesei ZU-02 c cht l li bp Mi 1. Chuyn ha sinh hc lignocellulosic biomass: ha sinh v tinh th ??? 2. Tng cng sn xut ethanol bng cch ln men rm thy phn bng cch s dng mt ging mi chuyn th ca stipitis Pichia 3. Ci tin mi sn xut cn t vt liu lignocellulose 4. Enzyme ethanologenensis chu nhit: trin vng tng lai trong vic sn xut ethanol th h th 2.

Mc lc
I. Tng quan v ngun nguyn liu ............................................................................... 3 1. Gii thiu v ngun ph thi li bp ...................................................................... 3 2. Cu trc li bp ....................................................................................................... 3 2.1. 2.2. 2.3. II. Cu trc ha hc ca lignin ............................................................................. 4 Bn cht ho hc ca cellulose ........................................................................ 5 Bn cht ho hc ca hemicellulose ................................................................ 5

Gii thiu v ngun enzyme ................................................................................... 6

1. Enzyme cellulase ...................................................................................................... 6 2. Ngun thu nhn enzyme cellulase .......................................................................... 9 2.1. 2.2. 2.3. 2.4. Chng Trichoderma reesi ................................................................................. 9 Chng Aspergillus niger ................................................................................ 10 Chng Aspergillus fumigatus ......................................................................... 10 Chng Thermomyces lanuginosus ................................................................. 11

3. Thu nhn enzyme cellulase t trichoderama reesi trn mi trng bn rn ... 12 III. S quy trnh chuyn ha vt liu lignocellulose thnh bioethanol .............. 14

1. Thuyt minh quy trnh .......................................................................................... 14 2. Cc hng nghin cu mi................................................................................... 18 2.1. Sn xut Bioethanol bng cc chng vi khun bin i gen ...................... 19

2.2. Sn xut Bioethanol bng phng php kt hp ng phn v ln men (SSF) .......................................................................................................................... 26 2.3. S dng kt hp cc chng vi khun nng cao nng sut thy phn ....... 33

I.

Tng quan v ngun nguyn liu 1. Gii thiu v ngun ph thi li bp - Cng vi xu hng pht trin mi ca ngnh cng nghip du kh v nhu cu s dng nhin liu ngy mt tng cao, t bit l nhin liu sch v r tin. Tn dng ngun ph thi nng nghip nh x da, rm r, bt giy v trong nghin cu ny l li bp ph thi to ra sn phm bioethanol, gip ci thin tnh hnh v gi c v nguy c cn kin ngun nguyn liu ha thch trn th gii. - Vit Nam bp l cy lng thc quan trng ng hng th hai sau cy la. Nm 1990 din tch cy bp trng trong c nc l 430 ngn ha, sn lng 670 ngn tn, nm 2000 714 ngn ha, sn lng l 2,183 triu tn, nm 2003 909.800 ha, sn lng 2,933 triu tn. Nh vy sn lng bp trong c nc l rt ln, sau khi thu hoch th ngi nng dn b i mt lng ln li tri bp. V th chng ta c th tn dng li bp sn xut bioethanol nh mt ngun nguyn liu l tng. 2. Cu trc li bp Li bp kh cng bao ngm nhiu loi m khc nhau nhn chung c th chia lm 4 vng : vng li, vng my, vng r v vng m cng.

Hnh 1.1 cu trc li bp: I-vng li, II- vng m cng, III- vng r, IV- vng my Li bp l mt dng vt liu lignocellulose bao gm cellulose, hemicellulose, lignin, khong (tro), phenolic ha tan, acid bo v cc hp cht khc (cc cht trch ly). Trong li bp c cha khong 32.3-45.6% cellulose, 39.8% hemicellulose ch yu l ng pentose v 6.7-13.9% lignin (c th thay i ty theo ging). Hm lng cellulose trong li bp tng i cao so vi cc ph liu nng nghip khc. Hm lng hemicellulose chim th hai trong li bp, tn ti trng thi v nh hnh, d thm nc v cn tr s tip

xc ca enzyme vi cellulose v vy cn phi loi b trong qu trnh x l, tuy nhin bn thn hemicellulose l mt polysaccharide nn c th tn dng ln men. Phn cn li trong li ng l lignin chim hm lng tng i thp v cn loi b tng kh nng tip xc ca enzyme vi cellulose. Bng 1.1: cc thnh phn lignocellulose trong li bp theo khi lng kh Thnh phn Cellulose Hemicellulose Lignin Khi lng kh (%) 35- 48 22- 30 15- 27

2.1. Cu trc ha hc ca lignin Lignin l mt loi polymer rt phc tp. Mi mt phn t lignin cha khong 10000 n v. Chnh v c s dao ng ln ny nn hin nay cha c mt phng php tht chnh xc xc nh thnh phn lignin. Trong cu trc thc vt, lignin thng l mt vt cht lp y vo khong trng gia cellulose v hemicellulose. Do chng to ra lin kt cng ha tr vi hemicellulose v l mt cu ni gia cc b si vi nhau, lm tng tnh cht c hc cho t bo thc vt. Lignin l carbohydrate duy nht c tnh k nc, n lm cn tr s thm nc vo t bo. Lignin c to ra bi 3 thnh phn c bn l p-Coumaryl, Sinapyl v Conifery. Cu trc lignin rt d b thay i trong iu kin nhit cao v PH thp. nhit cao, lignin thng to ra khi ring bit, tch ra khi cu trc ca cellulose v hemicellulose. Chng cng tn ti dng nh hnh v v nh hnh. Lignin l mt polymer rt bn.

Hnh 1.2 Cu trc ca lignin

2.2. Bn cht ho hc ca cellulose Cellulose l mt polymer t nhin, c cu to bi D-glucose, cellobiose, arabinose, galactose, trong thnh phn chnh l glucose.

Hnh 1.3 Cu trc mch thng ca cellulose Trong cu trc ca cellulose, ngi ta thy c hai vng rt khc nhau. l vng kt tinh v vng v nh hnh. vng kt tinh, cc thnh phn to nn vi si cellulose thng kt hp cht ch vi nhau, to nn cu trc ng nht, c mt trt t xc nh, to ra cc b si. Cc b si ny c sp xp song song vi nhau. Cellulose vng ny c cng rt cao, khng n hi, enzym rt kh tc ng. vng v nh hnh, cc si cellulose khng sp xp theo mt trt t cht ch nh trn. Do lc tng tc gia cc thnh phn khng ng u. Vng v nh hnh cellulose d b cc enzym phn gii hn vng kt tinh.

Hnh 1.4 Cc lin kt trong cu trc khng gian ca cellulose 2.3. Bn cht ho hc ca hemicellulose Hemicellulose l mt polymer t nhin khng hon chnh trong qu trnh to ra cellulose. Hemicellose c cc thnh phn l xylose, mannose, arabinose, rhamnose.

Hnh 1.5 Cc n phn ca hemicellulose Hemicellulose c cu trc v nh hnh, phn nhnh v mch ngn. Chng c lc lin kt yu. Mt s c im chnh ca hemicellulose nh sau: Mch chnh ca hemicellulose c cu to t lin kt -(1,4). Xylose l thnh phn quan trng nht. So vi cellulose, hemicellulose l polymer c khi lng phn t thp hn. Thnh phn chim nhiu nht trong cu trc hemicellulose l xylan. Xylan li c cu thnh t 85- 90% l D-xylose, mt lng nh L-arabinose v mt t acid glucuronic. Nhm th ph bin nht l nhm acetyl O- lin kt vi v tr 2 hoc 3. Mch nhnh cu to t cc nhm n gin, thng thng l disaccharide hoc trisaccharide. S lin kt ca hemicellulose vi cc polysaccharide khc v vi lignin l nh cc mch nhnh ny. Cng v hemicellulose c mch nhnh nn tn ti dng v nh hnh v v th d b thy phn. II. Gii thiu v ngun enzyme 1. Enzyme cellulase Khi nim v cellulase Cellulase l mt loi enzyme, c th c sn xut t nm mc, vi khun hoc sinh vt n bo; c kh nng thy phn cellulose v c hemicellulose. K hiu l EC 3.2.1.4 Phn loi cellulase Mc d ph bin trn tri t, cellulose li rt bn vng v kh b ph v v cellulose c kt tinh cao, khng tan trong nc, c kh nng chng li cc qu trnh depolymer ha. Qu trnh thy phn cellulose to thnh glucose c thc hin nh s tc dng hip ng ca 3 enzyme khc nhau:

Endo-1,4--glucanases (EG) hay 1,4--D-glucan glucanohydrolases (EC 3.2.1.4), Exo-1,4--D-glucanases bao gm 1,4--D-glucan glucohydrolase (EC 3.2.1.74),

enzyme ny s tn cng ngu nhin vo cc c cht 1,4--glucan c tan v khng tan. enzyme ny c tc dng gii phng D-glucose t 1,4--D-glucan v thy phn chm Dcellobiose; ngoi ra cn c enzyme 1,4--D-glucan cellobiohydrolase (EC 3.2.1.91) (CBH), enzyme ny s gii phng cellobiose t 1,4--glucan. -D-glucosidase hay cn gi l -D-glucoside glucohydrolase (EC 3.2.1.21) c tc dng to thnh D-glucose t celobiose l cellodextrin, cng nh cc olygomer ca glucose.

Hnh 2.1 Tc dng ca tng enzyme trong cellulase Cu trc ca cellulase Trong nhng nm 1970, nh nhng pht trin trong lnh vc ha sinh v sinh hc phn t, ngi ta c th nghin cu c cu trc ca cellulase thng qua ging nm mc Trichoderma reesi. y l chng nm mc ph bin sn sinh ra enzyme cellulase. Vo cui nhng nm 1980, Abuja ngh cu trc bc 3 ca Trichoderma reesei CBH I (enzyme thy phn cellobiose I cellobiohydrolase I) v CBH II. Trong , enzyme gm: Trung tm xc tc (CD: catalytic domain) vi kch thc ln Trung tm to lin kt vi cellulose (CBD: cellulose binding domain) c kch thc nh hn. Nghin cu cu trc ca trung tm to lin kt vi cellulose CBD ca CBH I cho thy y l mt chui polypeptide gm c 36 amino acid v c mt mt th hin tnh cht k nc mnh. V mt l thuyt, c th kt lun rng CBD c vai tr quan trng trong vic n

nh s lin kt tm thi gia cellulase v b mt cellulose. Trn b mt cellulose c vng k nc l do s sp xp cht ch v do lin kt hydrogen mnh gia cc mch cellulose, gp phn ngn cn, khng cho cc phn t ln nh nc xm nhp vo cu trc ny. Chnh tng tc gia hai vng c tnh cht k nc ca cellulose v CBD m enzyme c lin kt vi cellulose. Cu ni peptide: c tc dng lin kt hai trung tm ny li vi nhau. i vi enzyme cellulase, cu ni peptide l mt vng i ng, c cu to bi cc amino acid serine, threonin v proline. i vi T. reesei CBH II, li protein c tc dng ph v cu trc vi si ca cellulose. Sn phm thy phn ca CHB I gi nguyn cu trc lp th ca C1(C cha nhm OH hemiacetal), trong khi CBH II to s nghch o cu hnh ca C1 thnh ng phn . Cellulase c ngun gc t cc ging nm mc khc cng nh t vi khun u c cu trc tng t. C ch xc tc ca cellulase T nhng nm 1954, Gilligan v Reese ch ra rng vic pha trn cellulase t cc ngun nm mc khc nhau c th lm tng lng ng kh to thnh trong qu trnh thy phn. T lc , nhiu nghin cu c tin hnh nhm m t qu trnh tc ng hip ng ca cc enzyme endo, exo cellulase v -glucosidase. S tc ng hip ng ca endo-exo c th ni l nhm mc ch to b mt tn cng mi cho exoglucanse. Enzyme exo thng tn cng vo nhng cellulose b ct mch (cellodextrin) c u l nhm kh (C cha OH hemiacetal) hoc khng kh. Trong khi , enzyme endo li tn cng ngu nhin vo gia mch cellulose, to thnh cc cellodextrin, l cc c cht thch hp ca enzyme exo. Exoglucanase c nhng loi enzyme tn cng c trng ty theo tnh cht kh c hay khng kh c ca nhm u mch. Tht vy, theo nhng nghin cu ca Teeri v cng s, nhm kh u mch ca cellodextrin s c nh hng vo trung tm xc tc ca CBH I. Trong khi , CBH II li u tin tn cng vo u khng c nhm kh ca cellodextrin. Ngoi ra, cng c nhiu nghin cu v s tc ng hip ng ca cc enzyme endo, exo c ngun gc nm mc v vi khun. Da trn cc nghin cu ny, enzyme celluase c th c pha trn vi cc thnh phn t cc ngun khc nhau nhm to c mt h enzyme c tc ng mong mun. Enzyme endo-cellulase tn cng ngu nhin vo mch cellulose nh to lin kt bng tng tc gia CBD vi cellulose, to thnh cc oligosaccharide. Enzyme exo cellulase tn cng vo cellulose v c oligomer t u ng kh v khng kh thng qua tng tc ca CBD vi cellulose, to thnh cellobiose, c glucose.

 -glucosidase tn cng cellobiose v oligosaccharide tan, to glucose.

Hnh 2.2 C ch tc ng hip ng ca cc enzyme 2. Ngun thu nhn enzyme cellulase Ngun thu enzyme cellulase ln nht hin nay l vi sinh vt (nm, vi khun). Nhiu loi nm nh Trichoderma, Penicillium, Aspergillus, v T. emersonii c th sn sinh ra mt s lng ln cellulase v hemicellulase ngoi bo. Vt liu lignocellulose b thy phn bng enzyme iu kin n ha (500C v PH ~ 5), cho php phn ct cellulose v hemicellulose mt cch hiu qu m khng hnh thnh nn cc sn phm ph c th c ch hot ng ca enzyme . 2.1. Trichoderma reesi : a s cc dng nm Trichoderma pht trin trong t c pH t 2.5 n 9.5. Pht trin tt pH 4.5- 6.5. Nhit Trichoderma pht trin ti u thng l 25-30oC. Mt vi dng pht trin tt 35oC. Mt s t pht trin c 40oC . Trichoderma c xem nh nhng nh my sn xut nhiu enzyme ngoi bo hiu qu. Chng c thng mi ha trong vic sn xut cc cellulase v cc enzyme khc phn hy cc polysaccharide phc tp. Trichoderma tit ra mt lng ln cc enzyme khc nhau, c kh nng phi hp phn hy tinh th cellulose. Trichoderma reesi sn sinh ra 5 enzyme endoglucanase, 2 exoglucanase v 1 cellobiase (-glucosidase).

Hnh 2.3 Trichoderma reesi 2.2. Aspergillus niger Thng c gi l mc en, si nm mu trng, nh bo t mu en. T si u tin chng phn nhnh to ra 2-4 nhnh nh ri mi n bo t. Chng to ra rt nhiu loi enzyme nh: amylase, invertase, maltase, protease, pectinase, glucoamylase, cellulase Nhiu vi sinh vt phn gii cellululose nhng ch c Trichoderma v Aspergillus c nghin cu k lng nht v h enz cellulase. Trichoderma reesi sn xut lng ln cellulase nhng hot tnh -glucosidase rt thp. Vic b sung -glucosidase tng hiu qu ng ha nhng chi ph enzyme cn c cn nhc. Hnh 2.4 Aspergillus niger 2.3. Chng Aspergillus fumigatus Trichoderma reesei c bit n vi h thng sn xut enzyme cellulase cho hiu qu cao, nhng vic sn xut -glucosidase th li rt thp. Tuy nhin -glucosidase th hin v tr quan trng trong cng tc thu phn cellulose bng cch chuyn i cc cellobiose thnh glucose. Bn cnh , cc chng loi nm mc Aspergillus cho hiu qu sn xut -glucosidase rt cao so vi Trichoderma reesei. T l do m cellulase bt u c sn xut t nhiu chng loi Aspergillus khc nhau. Aspergillus fumigatus c la chn v c s dng sn xut ra exoglucanase (EXG; EC 3.2.1.9) v

endoglucanase (EG; EC: 3.2.1.4) , CMcase,

-glucosidase (EG; EC: 32.1.21) v

xylanase (Xy; EC: 3.2.1.8). Hot ng ca Aspergillus fumigatus hiu qu nhit 35-450C, v nhit ti u l 400C trong khi i vi Aspergillus niger th nhit hot ng hiu qu l 340C. Tuy nhin, nhit t 30-450C l khong nhit ti u cho hot tnh cellulase v xylanase sn xut t nm mc. Khong pH hot ng ca chng ny l t 5,0-7,0. Tuy nhin pH 6,0 l pH ti u cho hot ng ca enzyme sn xut t Aspergillus fumigatus. Bng 2.1 Hot tnh enzyme cellulase t mt s chng Aspergillus

2.4. Chng Thermomyces lanuginosus Chng Thermomyces lanuginosus c pht hin vi kh nng sn xut ra hemicellulase nhng chng cn c kh nng sn xut ra xylanase ngoi bo hot tnh cao. Cc enzyme ny m nhn vai tr thu phn vt liu hemicellulose. Hemicellulose chim khong 20% - 30% trong cu trc ca lignocellulose . Trong cc hemicellulase th xylanase t vi sinh vt c nghin cu rng ri nht xc tc thu phn xylan.

Hnh 2.5 Thermomyces lanuginosus

Thermomyces lanuginosus sinh sng ph bin trn tri t v c nhiu nc c lp t ngun thc vt mc nt. Chng l chng nm mc a nhit, pht trin ti a nhit 600C v ti thiu l 200C v nhit tng trng ti u l 500C. So vi cc loi nm a nhit khc, nhit pht trin ti a l 500C v ti thiu l nh hn 200C v nhit ti u cho s tng trng l 370C. Gi tr pH ti u cho s tng trng ca chng Thermomyces lanuginosus l 6.5 . 3. Thu nhn enzyme cellulase t trichoderama reesi trn mi trng bn rn Nm si: Chng T. reesei VTT-D-80133 nhn c t bo tng ging Roal Oy, Phn Lan. C cht: B ma v cm m. Ln men bn rn: xc nh thnh phn mi trng v cc iu kin ti u cho s sinh trng v sinh tng hp enzym, nm si c nui trn mi trng c t l b ma v cm m khc nhau (7:3, 6:4, 5:5, 4:6 v 3:7), m mi trng (t 50-70%), nng dinh dng (da theo mi trng Mandel, x1-x8 hay 1-8 ln) v thi gian nui cy t 2-12 ngy. Trch ly enzym: Cho 45 ml dung dch m Na-acetate 50 mM pH 5 vo 5 g canh trng, lc trn my lc vi tc 150 vng/pht trong 5 pht, lc thu dch. em ta dch lc bng cn 960c lm lnh trc. Thu kt ta v ha li vi cng th tch dung dch m. Xc nh hot tnh cc enzym: CMCase theo phng php ca Cng ty Shin Nihon Nht Bn vi c cht l CMC 1%; FPU theo phng php ca Hng Biopract GmbH c, vi c cht l giy lc Whatman no. 1; Xylanase theo phng php ca t chc EDC (Enzym Development Corporation, M), vi c cht l xylan 1%; -amylase theo phng php ca Hng Amano - Nht Bn, vi c cht l tinh bt 1%; Protease theo phng php ca Cng Ty Amano - Nht Bn, vi c cht l casein 1,5%. Xc nh hm lng protein: Theo phng php Bradford s dng bovine serum albumin nh protein chun. Thy phn giy: cho dch enzym cellulase (5 FPU/ml) vo giy xay nh (10%) 50 C, pH 5 trong 24 gi. Hiu sut (%) = lng ng kh (g)*0,9*(100/lng giy in (g)) in di protein: Sodium Dodecyl Sulfate Polyacrylamide gel (SDS-PAGE) c thc hin trn gel ng cha 10% (w/v) polyacrylamide. Ti u ha thnh phn mi trng bng phng php quy hoch thc nghim:
0

T l BM:CM, m ban u, nng dung dch dinh dng v thi gian nui cy l 4 yu t c nh hng r rt n kh nng sinh tng hp cellulase ca T. reesei VTT-D80133 nn c chn ti u ha theo phng php quy hoch thc nghim. KT QA V THO LUN Kt qa ti u ha thnh phn mi trng v cc iu kin nui cy. Cc kt qa th thim trc y, chng ti xc nh c thnh phn mi trng c s cho chng T. reesei VTT-D-80133 sinh ra cellulase theo phng php ti u ha c in: t l BM:CM (4:6), m ban u 54%, 5 ln nng dinh dng, thi gian nui 7 ngy, t l ging 6x106 bo t/g mi trng, hot tnh cellulase t c l 251,43 IU/g. Tuy nhin, thnh phn mi trng v cc iu kin nui cy mi ch c nghin cu nh hng mc ring r. Trong nm yu t trn th bn yu t l t l BM:CM, m ban u, nng dinh dng v thi gian nui cy c nh hng ng k n kh nng sinh tng hp cellulase ca T. reesei VTT-D-80133 nn c chn nghin cu ti u ha theo phng php qui hoch thc nghim. Qui hoch c thc hin vi ma trn y vi s th nghim N = 24 = 16 Kt qu quy hoch thc nghim nh sau: Mi trng ti u cho T. reesei VTT-D-80133 sinh ra cellulase l: t l BM:CM (7:3), 8 ln nng dinh dng, m ban u 60 %, thi gian nui cy 7 ngy. Hot tnh CMCase v FPU tng ng l: 280,63 IU/g v 5 FPU/g; thp hn 3,2 v 37 ln so vi Amano T. Ngoi cellulase, canh trng cn cha -amylase 368,75 UI/g, protease 12,43 UI/g v xylanase 10073,25 BXU/g. Dch enzym cellulase ca T. reesei VTT-D-80133 vi hot lc 5 FPU/ml, c kh nng ng ha khong 20% giy in qua s dng; dch ng ha cha 23,62 mg ng kh/ml c th c s dng ln men ethanol hoc ln men sn xut cc sn phm c gi tr.

III.

S quy trnh chuyn ha vt liu lignocellulose thnh bioethanol

Hnh 3.1 S chuyn ha sinh khi lignocellulose thnh nhin liu sinh hc 1. Thuyt minh quy trnh Bc 1. Tin x l: Giai on tin x l l cn thit nng cao hiu qu qu trnh thy phn cellulose thnh ng. Cc yu t nh hng n qu trnh thy phn cellulose bao gm xp ca vt liu, kch thc vi si cellulose, v s c mt ca lignin, hemicellulose trong vt liu . S hin din ca lignin v hemicellulose lm cho hot ng ca axit v enzyme cellulase tr nn kh khn hn, do hiu xut ca qu trnh thy phn s thp. Tin x l l cn thit thay i cu trc v kch thc ca sinh khi, cng nh thnh phn ha hc ca n, sao cho qu trnh thy phn cc hydrocarbon thnh cc loi ng n din ra nhanh chng v t hiu qu cao. Qu trnh thy phn s t hiu qu cao bng vic loi b lignin v hemicellulose, gim kch thc vi si cellulose, tng cng xp thng qua qu trnh tin x l .

Hnh 3.2 Tin x l lignocellulose trong sn xut bioethanol .

Cc phng php thc hin Tin x l bng phng php vt l (phng php c hc) Tin x l bng phng php ha hc (acid hoc bazo) Tin x l bng ozone Tin x l bng dung mi hu c Tin x l bng cc cht lng ion ( Ionic liquids ILs) Tin x l bng phng php ha l Tin x l bng phng php sinh hc Bc 2. Thy phn (hay cn gi l qu trnh ng ha). Bn cht ca qu trnh: (C5H10O5)n + nH2O Thy phn bng acid Thy phn bng enzyme: Cc mt xch ca cellulose c th b phn ct thnh cc phn t ng glucose ring l bng enzyme cellulase. Enzyme cellulase l mt phc h enzyme c tc dng thu phn cellulose thng qua vic thu phn lin kt 1,4--glucoside trong cellulose to ra sn phm glucose. Bc 3. Ln men: bn cht qu trnh ln men Trong qu trnh ln men, cc sn phm ca qu trnh thy phn bao gm ng hexose (glucose, mannose v galactose) v pentose (xylose v arabinose) s c ln men thnh ethanol. Trong s cc sn phm ca qu trnh thy phn, glucose l phong ph nht, tip sau l ng xylose hoc mannose v mt t cc ng khc. Phng trnh toam tt ca qu trnh ln men ru c biu din nh sau: C6H12O6 2C2H5OH + 2CO2 + Q Saccharomyces cerevisiae l vi sinh vt truyn thng c s dng ln men glucose thnh ethanol. S. cerevisiae c rt nhiu u im nh ph bintrong t nhin, t l ln men cao v lng ethanol to thnh cao. C ch ca qu trnh ln men ethanol t gucose nh sau: nC6H12O6

Hnh 3.3 C ch ca qu trnh ln men ethanol

c im v cu to ca nm men Saccharomyces cerevisiae - Gii: Fungi - Ngnh: Ascomycota - Phn ngnh: Saccharomycotina - Lp: Saccharomycetes - B: Saccharomycetables - H: Saccharomycetaceae - Chi: Saccharomyces - Loi: Saccharomyces cerevisiae Hnh 3.4 Saccharomyces cerevisiae Hnh dng v kch thc t bo nm men : Saccharomyces cerevisiae l cc t bo hnh ellipse, c chiu di t 9 n 10 m v chiu rng t 2 n 7 m. T bo nm men thng c kch thc ln gp 5- 10 ln t bo vi khun. Kch thc ca t bo nm men thay i theo iu kin nui cy, theo tui sinh l. V d : Saccharomyces cerevisiae c hnh bu dc nu mi trng giu cht dinh dng, cn trong iu kin ym kh nm men c hnh trn, iu kin hiu kh t bo li ko di hn. Nm men th hin hu ht cc cu trc v chc nng ca t bo nhn chun, v c s dng nh l mt m hnh chung cho cc t bo nhn chun sinh hc Cu to t bo nm men Nm men l sinh vt n bo. So snh cu to t bo nm men vi vi khun ta thy c s tin ha nhy vt t nhn s n nhn chun. Cng vi s tin ha v nhn v c ch phn chia nhn ( nhn c mng, c cc nhim st th), t bo nhn chun xut hin nhiu th khng thy t bo nhn s nh ti th; lc lp Hnh 3.5 Cu to t bo nm men T bo nm men cng nh nhiu loi t bo khc c cu to ch yu t cc thnh phn c bn nh : thnh t bo, mng nguyn sinh cht, nhn, cc c quan con khc ( khng bo, ty th, ribosome).

Bc 4. tch chit v tinh ch: s dng phng php chng ct 2. Cc hng nghin cu mi

Hnh 3.6 Quy trnh chuyn i sinh khi lignocellulose thnh ethanol Qu trnh chuyn i sinh khi lignocellulose thnh ethanol, bao gm cc bc chnh: Thy phn v ln men c th tin hnh c lp (SHF, c th hin bng mi tn nt t), hoc tin hnh ng thi (SSF). Trong qu trnh sinh hc hp nht (CBP), tt c cc bc bin i s c tch hp vo trong mt bc duy nht bng cch s dng mt hoc nhiu loi vi sinh vt. Cc k thut khc nhau nh gy t bin gen, ng nui cy (co-culturing) v biu hin gen (heterologous gene) c s dng ci thin kh nng s dng ng ca vi sinh vt cng nh ci thin hot tnh v s n nh ca enzyme thy phn c ngun gc t nm nng cao sn lng ethanol tng th.

2.1. Sn xut Bioethanol bng cc chng vi khun bin i gen Sn xut ethanol bng cch thy phn li bp nh Escherichia coli KO11 Thy phn li ng vi sn phm l xylose l ng chim u th, c ln men thnh ethanol bng cch ti t hp Escherichia coli KO11. Khi s dng ngun dinh dng l ng glucose qu trnh ln men hon ton din ra trong 163h v to thnh 0.5g ethanol/g ng. Khi ngun cacbon l xylose, nng sut ethanol gim, nhng qu trnh ln men din ra nhanh hn (113 h). S thy phn c cha 72,0 g / l xylose v b sung vi cm go 20,0 g / l d dng ln men, sn xut 36,0 g / l ethanol trong vng 70 gi. Khi s dng mt t bo cao hn trong qu trnh thy phn khng lm tng hiu sut thy phn. Bi bo ny m t qu trnh cng nghip kt hp ln men ng pentose bng E. coli v ln men ng hexose bng nm men saccharomyces. bc u tin, E. coli ln men dung dch c cha 85.0g/l xylose, sn xut ra 40.0g/l ethanol trong 94h. sau , nm men bnh m v ng sucrose (150.0g.l) c thm vo hn hp ln men. Kt qu, sau 8h nng ethanol t 104.0g/l. Qu trnh ln men hai giai on ny ang c quan tm nhiu v hiu xut sn xut ethanol ln. Cc chng trnh sn xut ru Brazil l mt v d ni bt cho ngun nng lng ti to thay th cho nhin liu ha thch. Theo , vo nm 1985, s lng xe s dng ru ngm nc chim n 90% th trng xe mi. hin nay, ru khan c s dng nh mt ph gia cho xng du vi t l pha trn l 22-24%. Cc ngnh cng nghip nhin liu ethanol ang phi i u vi nhiu kh khn, nhng tng lai ca n cn nhiu ha hn. nguyn liu cho sn xut ethanol quan trng nht brazil l cy ma. Ngoi ra, d lng lignocellulose l rt phong ph phc v cho vic sn xut ethanol bng cch ln men cc loi ng pentose s dng enzyme vi sinh vt, song song vi qu trnh sn xut t nm men thng thng. Trong qu trnh tin x l lignocellulose th hemixenluloza l thnh phn d b thy phn nht cho ra pentoses v hexoses. S chuyn i cng su sc cng lm tng hiu qu v gim chi ph cho qu trnh sn xut. C th s dng nhiu phng php nng cao nng sut sn xut, nhiu tc gi ch ra rng chng E.coli bin i gen c kh nng chuyn ha pentoses v hexoses thnh ethanol vi hiu sut chuyn i ti a gn bng hiu sut l thuyt. Nghin cu ny kim tra kh nng ca chng E. coli KO11 sn xut ethanol t li ng. Nghin cu kh nng ci thin nng sut sn xut bng cch s dng kt hp gii c thy phn vi vi sinh vt thch hp hoc vi mt t bo cao. Cm go c th nghim nh mt ngun dinh dng b sung.

Vt liu v phng php Vi sinh vt: Chng E. coli K11 c s dng. c nui cy trn thch a cha mi trng LB c b sung 20g/l xylose v 40mg/l chloramphenicol v 300C trong 24h. cy truyn hng ngy. Chun b ging cho ln men: Khun lc c cy vo bnh erlen 250ml cha 50ml mi trng LB lng c b sung glucose hoc xylose 40g/l, nui nhit 300C trong 6h trn bnh lc. trc khi ln men pha long dch trn 10 ln t nng vi sinh vt mong mun. th nghim ci tin nng sut vi bnh erlen cha ht thy tinh v vch ngn. Chun b li ng Chun b dch ln men: ngm 100g li ng +trong 600ml acid sulfuric 1% (v/v) trong 24h. Tch b ra khi dung dch, b ng c hp 1200C trong 40 pht. Dch lng em lc, un nng n 800C, thm CaO chnh pH=10 v b sung N2S 1g/l. lm ngui v nhit phng, ly tm v chnh pH v 7 bng acid sulfuric. i vi th nghim ln men, dch thy phn c b sung tryptone 10g/l v chit xut nm men 5g/l. ngoi ra ngun dinh dng cm go 20g/l cng c th nghim. cm go c chun b bng cch: thm 1000ml nc ct vo 200g cm go, hp 1270C trong 15 pht sau ly tm thu dch ni. Th nghim ln men Tt c cc th nghim c tin hnh 300C, trong bnh lc 100ml vi 45ml dung dch thy phn li ng. Bnh c bt kn bng nt cao su c khoan l ly mu ng thi kh CO2 thot ra. Hm lng ng c chuyn ha v nng ethanol c xc nh bi phng php HPLC, s dng bio-rad HPX-87h. Tnh ton cc thng s ln men Nng sut l thuyt ti a ca ethanol t xylose l 0,51 g ethanol / g ng. Nng sut th tch (QP) c tnh bng cch chia nng ethanol ti a bng thi gian cn thit t c nng (g/lh). Sn lng ethanol (YP / S) c tnh ton nh nng ti a ca ethanol sn xut chia cho nng ng ban u hin din trong mi trng (g ethanol / g ng). Kt qu v tho lun Cc ph thi nng nghim quan trng nh b ma hoc li ng c th c s dng lm nguyn liu m rng quy trnh sn xut bioethanol brazil. Sn xut ethanol nng sut cao bng cch s dng chng E. coli KO11 ln men dch thy phn hemicellulosic giu pentose kt hp vi quy trnh sn xut ethanol t hexoses bi nm men thng thng.

Vic chuyn i lignocellulose thnh ethanol th rt phc tp v tri qua nhiu bc nh: tin x l, gii c, b sung cc cht dinh dng. tin x l bng acid l phng php hiu qu gii phng monomer ng t hemixenluloza nhng li to ra cc cht c ch cho qu trnh ln men. V th cn phi tin hnh kh c bng cch km hm hoc loi b cc cht c hi trn. Cng vi khu kh c, b sung dich dng cng l mt bc quan trng chim mt phn ng k chi ph sn xut. trong nghin cu ny, hemicellulosic thy phn t li ng c s dng lm nguyn liu sn xut ethanol bng E. coli KO11. Chng vi sinh vt ny c kh nng chu mi trng acid do qu trnh thy phn cn st li cng nh chu c cc cht c sinh ra, tin hnh th nghim s dng mt vi sinh vt cao ci thin nng xut ethanol. Overlimed v trung ha dch ln men bng cch thm tryptone v chit xut nm men chng ti t hp c th pht trin tt trn mi trng ny. Khi nui cy vi sinh vt trn mi trng LB c cha glucose (hnh 1), s ln men c cht hon tt trong 163h t nng ethanol ti a l 39.7g/l tng ng vi 0.5g ethanol/g ng. khi E. coli c nui cy trn mi trng LB c b sung xylose nh ngun ng duy nht c biu din trn hnh 2. Sn lng ethanol thp hn nhng s ln men din ra nhanh hn, trong vng 113h. Khi nui cy E. coli trn mi trng dinh dng xylose s c nh hng tch cc n qu trnh ln men t dch thy phn giu pentose.

Hnh 3.7 Ln men thy phn hemixenluloza t ng bp E.coli KO11. Chng vi sinh vt pht trin trn mi trng LB b sung vi glucose. ng tiu th sn xut ethanol.

Hnh 3.8 Ln men thy phn hemixenluloza t ng bp E.coli KO11. Chng ging trc y c mui cy trn trung bnh LB b sung vi xylose. ng tiu th sn xut ethanol. Dch thy phn t li ng c th nghim kim chng hiu qu ca vic b sung cm go cung cp vitamin. Kt qu cho thy vic b sung cm go mang li hiu qu cao. nng ethanol ti a t c trong vng 69h v nng sut l gn 0.5g ethanol/g ng. nu khng b sung cm go, chng ti t hp chuyn ha xylose chm hn, hn na nng sut ethanol gim xung cn 0.45g ethanol/g ng (bng 3.1). Bng 3.1 Hm lng xylose c chuyn ha v nng ethanol trong dung dch

 kim tra kh nng ci thin qu trnh ln men bng cch s dng nng t bo cao hn, dch nui cy c chun b bng cch chuyn mt khun lc vo bnh nui cy c hoc khng c vch ngn cha mi trng LB c b sung xylose. Hn na, cc bnh th nghim c kt hp vi vic b sung cc ht thy tinh tng cng kh nng khuy o t lm tng s ng nht ca dung dch trong bnh th nghim. bnh khng c vch ngn: nng t bo t 2g/l trong vng 5h v khng tng thm na. vi bnh c vch ngn v c b sung cc ht thy tinh nng t bo t 4.2g/l. s gia tng mt t bo c gii thch l do tng kh nng thong kh v t bo tip xc vi c cht hiu hn nh c vch ngn v ht thy tinh. (hnh 3)

Hnh 3.9 S pht trin ca E. coli KO11 trong mi trng LB, s dng bnh canh tc c hoc khng c vch ngn, v b sung cc ht thy tinh. Bnh m khng c vch ngn; bnh vi vch ngn, bnh vi vch ngn v b sung knh ht. Th nghim nh sau: hai bnh cha mi trng LB c b sung glucose vi nng 50g/l c cy ging vi mt khc nhau: Bnh 1: 2.0g/l. Bnh 2: 4.2g/l Kt qu bnh 1 t nng ethanol l 17.8g/l trong 43h. Bnh 2 lng ethanol sinh ra cao hn khong 40% so vi bnh 1. Mi trng LB b sung 42g/l xylose v dch thy phn li ng c cy ging vi t l nh trn cho kt qu nh sau: nng ethanol tng ng vi mi mt t bo c cy vo l

23g/l v 19.6g/l tng ng vi sn lng ethanol l 0.46g/g (g ethanol/g ng tiu th). Thi gian ln men cho c hai trng hp l 163h nhng kt qu l nng ethanol cui cng l 39,7 v 28,0 g/l tng ng vi nng t bo thp v cao (hnh 4).

Hnh 3.10 nh hng ca cy khc nhau v s ln men ca ng thy phn li ng hemixenluloza bi E. coli KO11.(I) cy nng 4,2 g / l tiu th ng, sn xut ethanol.(II) cy nng 2,0 g / l: tiu th ng sn xut ethanol. Dch ln men c cy vi mt ging l 2g/l cho sn lng ethanol cao hn so vi mt ging l 4.2g/l. y l kt qu ng ngc nhin v thng thng th nng t bo cao hn s cho nng sut cao hn. T ta kt lun rng vi sinh vt c th s dng ethanol hoc sn xut ethanol rt t trong iu kin mt t bo cao. Cui cng, chin lc ln men hai giai on c nghin cu, s dng c hai chng vi sinh vt l E. coli KO11 v nm men bnh m t c nng ethanol ti u. Trong bc u tin chng E. coli KO11 ln men dch thy phn li ng c b sung tryptone v chit xut nm men, nng ethanol ti a t c l 40g/l trng vng 94h tng ng vi sn lng l 0.47g ethanol/g ng hiu sut l 92% so vi l thuyt. Trong bc th hai, 100g/l men bnh m v 150g/l ng sucrose c thm vo dch ln men ngay khi bc mt kt thc. Sau 8h, ng sucrose c ln men hon ton thnh

ethanol bi nm men v nng ethanol cui cng tch ly trong mi trng l 107 g / l (Bng 3.2). Bng 3.2 ln men dch thy phn li bp bng cch s dng chng ti t hp E. coli KO11 v nm men (100 g/l) cng vi 150 g/l sucrose trong hai giai on ca qu trnh sn xut ethanol

E coli KO11 sn xut axit axetic, lactic v succinic vi s lng nh trong iu kin ym kh, nhng sau khi hon thnh bc u tin, pH ca mi trng l 6.0 v khng cn thit iu chnh n trc khi thm ng sucrose v men. R rng, s thy phn hemicellulosic trnh by mt kh nng m do s hin din ca cc hp cht hot ng nh b m hot ng ti pH dung dch, v s hin din ca E. coli khng nh hng n bc hai ca qu trnh. Hn na, khng cn thit phi loi b t bo vi khun trc khi b sung nm men. Vi nng ethanol cui cng t 10%, cc t bo vi khun cht v khng tc ng xu n mi trng. Qu trnh ln men hai giai on bo co trn c th c s dng ti Brazil, vi cc chng ti t hp E. coli KO11 thc hin qu trnh ln men pentose kt hp ln men thng thng chuyn ha hexose. Hin nay, tng sn lng ethanol u xut pht t s ln men ca nc ma bng nm men. Nu tt c cc loi ng ln men tm thy trong hemixenluloza c th c phc hi v s dng, n s thm mt lng ng k c cht sn xut ethanol tng nng sut sn xut ethanol trong nc.Bn cnh li ng, b ma phi c xem nh l mt nguyn vt liu quan trng tim nng sn xut ethanol.Sucrose c la chn l nn bt u t bc th hai bi v n l phong ph Brazil v cc kh

nng mi cho s dng ca n phi c nghin cu. chng ta c th thy c li tht khi s dng kt hp hai chng vi sinh vt trong cng mt qu trnh ln men duy nht. E. coli KO11 c th chuyn i cch hiu qu ng pentoses thnh ethanol v nm men chuyn ha hiu xut cao ng hexoses, ethanol c bn cao v thi gian tn ti lu di trong cc thit b ln men. Qu trnh ln men hai bc ny yu cu thit k mt nh my c th, trong qu trnh ln men thy phn hemicellulosic bi E. coli xy ra trong mt bnh c lp. sucrose v nm men c thm vo sau bc ln men ny. Qu trnh ny c th c kt hp vi mc tiu ti a ha nng sut, lm cho vic s dng ngun ti nguyn c trit nht. 2.2. Sn xut Bioethanol bng phng php kt hp ng phn v ln men (SSF) Gii thiu Ng l loi ng cc di do nht c trng Ghana v i km vi s lng ln 30% dch li ng c hnh thnh. Dch ny hin nay ang c tn dng sn xut ethanol sinh hc. Aspergillus niger phn lp t t cc trang tri Ejura c s dng thy phn li ng thnh cc ng n gin. Dch lc thu c t nc li ng c ln men bi A. Niger nh enzyme th trong cc th nghim ti u ha trn huyn ph bt li ng trong 50 mM citrate m pH 5.0. Nhit , pH v nng c cht ti u cho s ng ha l 40oC, 4.0 v 6%. Saccharomyces cerevisae c thm vo dch lc A. niger gy ra qu trnh ln men ca li ng. Nng ethanol cao nht l 0.64 g/l c ghi nhn trong sut 24h ln men. Mt s kh khn khi sn xut ethanol t vt liu lignocellulose: V mt k thut: Nhiu kin cho rng nng lng sn xut nhin liu ln hn nng lng tiu th c t n. Ngoi ra, bioethanol l mt nhin liu km hiu qu hn xng, ch cha nng lng bng 70% so vi xng. Mt khc, s dng c xng c nng ethanol cao, mt s xe cn phi ci bin. Bioethanol pht thi nhiu kh NOx v n mn kim loi hn xng thng thng. Hin nay, cc nh khoa hc ang nghin cu tm cch hn ch cc nhc im ny bng cc bin php cng ngh nh trn thm cc ph gia chng n mn. Tuy nhin, trc mt, cc nhc im ny c th chp nhn c v phng din c hc v mi trng khi pha trn vi t l thp. Bioethanol c p sut hi thp, v vy s gy kh khn cho vic khi ng lnh ng c khi nhit d di 150C. h tr trong giai on ny th cn phi c mt h thng khi ng lnh

2.2.1 Vt liu v phng php Cellulase sn xut bi A. Niger. Cellulase c sn xut trong dch li ng (CBB). CBB l mt mi trng Mandel sa i c m t bi Jeffries (1987) v bao gm nhng thnh phn sau cho mi lt: (NH4)2SO4

1.4 g, KH2PO4 2.04 g, CaCl2 0.3 g, MgSO4.7H2O 0.3 g, axit citric 0.25 g, Tween 80

2 ml, Avicel cellulose 10 g, nm men 1 g v dung dch kim loi 1 ml. Dung dch kim loi bao gm ha cht sau y cho mi 500 ml: FeSO4 2.55 g, MnSO4.H2O 0.93 g, ZnSO4.H20 1.78 g, Co(NO3)2.6H2O 1.25 g, v Conc. HCl 5 ml. 250 ml CBB c cy 3.02107 bo t ca A. niger v trn my lc (mi trng G24, New Brumswick Co. USA) 120 rpm ti 25oC trong 6 ngy. Canh trng c ly tm 10000 vng trong 10ph v phn ni trn c s dng nh enzyme th trong cc th nghim ng ha. xc nh hot ng ca enzyme trong giai on ny, 5 ml c thu thp ti khong thi gian 24 gi. 2.2.2 Cc th nghim ti u Phng php ca Baig (2004) c cng nhn. 1% huyn ph li ng c chun b bng cch huyn ph ha 10 g c cht trong 1 lt m citrate 50 mM pH 5.5 v hp 121oC trong 20 ph. 15 ml huyn ph c cht c cho vo Erlenmeyer thm nc ti 100 ml v 5 ml ca dch lc thu c t dch nui cy A. niger c thm vo mi bnh. Qu trnh ng ha c thc hin trong mt t lc 27 2C trong 24 gi. Huyn ph sau ly tm (2500 vng, 15 pht) c kho nghim lng ng tng bng phng php DNS. Cc loi ng gii phng u c th hin tng ng vi glucose. Nhit ti u ca qu trnh ng ha c xc nh bng hn hp phn ng nhng khong nhit khc nhau t 25-60C. pH ti u c xc nh bng cch iu chnh pH ca hn hp phn ng t 3,5 n 6,5. Nng c cht ti u c xc nh bng cch chun b huyn ph c cht t 1-6%. 2.2.3 Chun b dch cy nm men Nm men cy c chun b nh m t ca Scholar v Benedikte (1999) v Suh (2007). Mt gam nm men kh c mua t ch Kumasi c pht trin trn mi trng Yeast Peptone Dextrose (YPD) 30oC trong 48 h c hot ha v kim tra tp nhim. Mt khun lc nm men c chuyn n 100 ml YPD 5% nhit phng trn mt my lc 130 rpm trong 48 h. 7 ml canh trng c ly tm 4500 rpm trong 5 pht. Phn rn c chit ra bnh, v c ra trong 10 ml nc ct v trng hai ln, ly tm v chit phn rn ra bnh. Tip tc huyn ph li vi 50 mM b m citrate theo t l 1/10 lm vic cho mi bnh v c s dng cy.

2.2.4 ng ha v ln men ng thi (SSF) SSF c thc hin nh m t ca Dowe v McMillian (2008). 6 g bt li ng, 1.0 g dch chit nm men v 2.0 g peptone c cho vo mi bnh Erlenmeyer 250 ml, thm m citrate 50 mM, pH 5.0 vo to thnh 80 ml huyn dch v mi erlen c lp t hai l c gn silicone. Cc bnh v cc dng c km theo c hp 121oC trong 20 pht. Sau khi lm mt v nhit phng, 10 ml dch lc A. niger v 10 ml nm men cy cng c a vo mi bnh. Mi ng dn v trng c c nh vo trong mt l ca bnh v thit b kha ln men c c nh vi ng trong l khc ca nt bnh. Cc bnh c nhit phng trn my lc 110 rpm trong 24 h. 5 ml dch c rt ra t cc bnh 4, 6, 8, 12 v 24 gi a vo ng dn v trng. ng dn c x l sau mi ln ly mu. Mu c ly tm 10000 vng trong 10 pht v dch ni gi lnh phn tch sc k kh cho ethanol. 2.2.5 Xc nh nng ethanol bng sc k kh???? Nng ethanol c xc nh bng cch s dng mt Perkin Elmer, Autosystem XL, sc k kh (M) c trang b vi mt my d ion ha ngn la (FID), cng vi mt my ghi Yokogawa Bt 3021. Mt chromopak K 80/100 ct CRS c s dng. T l dng chy ca kh vn chuyn, N2, 42 ml / pht. H2 v khng kh nhin liu s dng. Nhit l nng, nhit vi phun v my d nhit l 130, 200 v 200oC. Khi lng tim 1 ml v thi gian lu gi l 8,5 pht. Xc nh v quantifycation c da trn so snh trc tip ca cc phn ng sc k kh tiu chun ethanol. Tt c cc th nghim c t trong thit k ngu nhin hon chnh v tng iu tr c th nghim trong ba ln. Phn tch ANOVA c tin hnh vi phin bn beta 7,5 Assistat (Tr l thng k, 2008). th c v vi Microsoft Excel. KT QU V THO LUN Mt A.niger l loi nm tt nht nm sn xut cellulolytic phn lp t t ly mu t cc trang tri Ejura c nui cy trn CBB trong 6 ngy trn bnh nui cy lc ch 120 rpm. Aliquots ca 5 ml c ly mu ti 24 gi khong thi gian v qua kho nghim cho cc hot ng enzyme. Hnh 1 minh ha cc hot ng enzyme trong thi gian 144 gi. C s gia tng kh nng hot ng ca enzyme t 24 n 144 h sau khi nui cy. Xenlulaza l enzyme cm ng v nng ca n tng ln cng vi s tng sinh khi ca nm trong thi gian nui cy.

Hnh 3.11 Hot tnh enzyme ca A. niger trn mi trng CBB trong 6 ngy ng ha ca bt ht bp trong 24 gi minh ha trong Hnh 2. C ng phn tng t 0 n 24 h. S gia tng dc ln n 9 h (giai on 1) hn l t 9 n 24 h (giai on 2). Vic lm chm tc thu phn phi l do hot ng ca cc enzym b chm li bi cc chng ngi vt cn tr con ng ca mnh hoc thua l trong hot ng v / hoc chuyn ha lm cho h km hiu qu (Yang et al, 2006).

Hnh 3.12 Hm lng glucose theo thi gian ng phn

Nhng nh hng ca nng cht nn, nhit v pH trn pht hnh gim ng cng c theo di. T l ng phn t l thun vi nng cht nn ln n nng cht nn ti u. iu ny l do va chm ngu nhin gia c cht v cc trung tm hot ng enzyme xy ra hn thng xuyn. ng ha tng theo nng cht nn nh trong hnh 3.

Hnh 3.13 nh hng ca nng c cht n qu trnh ng phn C c gia tng trong vic lm gim ng vi tng nng cht nn. Nng glucose trung bnh cao nht 3,1105 mg / ml c ghi nhn nng cht nn l 6% v khc bit ng k (p <0,05)???. Nng c cht 1% cho hiu sut ng phn thp nht. Nng glucose 6% cht nn nng cao hn v khc bit ng k t 5 nng cht nn%, cho thy nng c cht ti u l 6%

Hnh 3.14 nh hng ca nhit ln qu trnh ng ha Nhit c tc dng phc tp v hot ng ca enzyme v do ng phn. N nh hng n tc ca cc phn t; nng lng kch hot phn ng xc tc v n nh nhit ca enzym. Ni chung ng phn tng vi nhit ti u sau n suy gim. S gia tng nhit l do s gia tng tng ng trong lnh vc nng lng ng hc v suy gim sau khi bin tnh enzyme do ti u. nh hng ca nhit trn ng phn l hin th trn hnh 4. ng ha tng t 25oC n mc ti a 40oC sau gim ln n 60oC. Nhit (40oC) cng c ghi nhn khi tin hnh th nghim trn cht thi x da v mn ca. S thay i ang k hm lng ng to ra khi chuyn t nhit 50-600C chng t enzyme cellulase rt nhy vi nhit .

Hnh 3.15 nh hng ca pH ln qu trnh ng ha pH ca mt dung dch c tc dng v c cu v hot ng ca cc enzym v do ng phn.Enzym l nhng phn t lng tnh c cha mt s lng ln cc axit v cc nhm c bn, ch yu nm trn b mt ca chng.Nh vy pH nh hng n phn ng ca cc nhm hot ng xc tc. Hnh 5 minh ha cc nh hng ca pH n hm lng ng t c cht.ng ha tng t pH 3,5 n ti a l 4.0 sau n gim ln n 6,5.ng phn cao nht m l khc bit ng k (p <0,05) c ghi li pH 4.0. pH thp lin quan cung cp iu kin c tnh axit trong ngn chn nhim vi khun trong qu trnh ln men. Hnh 6 cho thy xu hng sn xut ethanol trn 24h thi gian 25oC v 110 rpm. SSF c thc hin vi10 ml dch lc ca A. niger. Ethanol nng caqu trnh ln men c xc nh ti 4, 6, 8, 12 v 24 gi sau khi cy. Nng ethanol trung bnh l0,4326 g / l. C gia tng sn xut ethanol trn giai on. C th c tng ln trong ng phntrong giai on ng c sn S. cerevisaecho ln men. Olofsson v cng s al bo co rng enzyme thy phn ca cc phn rn ln kim sot t l tng sn xut ethanol trong SSF.Ngoi ra nng ethanol v cc sn phm khc trong nc dng c th khng t n mc c ch ca men trong giai on ny. Nng cao nht 0,642 g / l c ghi nhn ti 24 h. So snh n nhngcng trnh vn hc l kh khn bi v nng ethanol khng c trch dn v chng khc nhau hoc trong loi tin x l nu c v gii c, nng cht nn, cng thng qu trnh ln men, nhit hoc ofoperation ch nh hng n nng ethanol cui cng trong cng vic bi Olofsson v cng s al.

Hnh 3.16 S hnh thnh ethanol theo thi gian 2.3. S dng kt hp cc chng vi khun nng cao nng sut thy phn Tm tt Cc ch phm enzyme thy phn li ng v ln men ethanol t cellulose c nghin

cu. Sau khi li ng c tin x l vi H2SO4 1% ti 108oC trong 3 gi, d lng cellulose c thy phn bi enzyme t Trichoderma reesei ZU-02 van lng thy phn l 67,5%. Hot tnh thp cellobiase trong T. reesei cellulase hn ch vic chuyn ha cellobiose thnh glucose, v lm tch t cellobiose gy ra c ch phn hi nghim trng n hot ng ca -1,4-endoglucanase v -1,4-exoglucanase trong h thng cellulase. B sung cellobiase t Aspergillus niger ZU-07 lm gim ng k s c ch gy ra bi cellobiose, v sn lng thy phn c ci thin n 83,9% vi hot tnh cellobiase nng cao t 6.5CBU/g c cht. Qu trnh thy phn fed-batch bt u vi mt m thy phn c nng c cht 100 g/l, vi d lng cellulose c thm vo lc 6h v 12h c c lng c cht cui cng l 200 g/l. Sau 60h phn ng, nng ng gim t 116,3 g/l vi lng thy phn 79,5%. Ngoi ra qu trnh ln men thy phn cellulose c cha 95,3 g/l glucose c thc hin bng cch s dng nm men Saccharomyces cerevisiae 316, v 45,7 gl ethanol c thu nhn trong vng 18 gi. Cc kt qu nghin cu c ngha trong sn xut ethanol nhin liu t ph phm nng nghip thay v tinh bt ng cc.

Gii thiu Khi ngun nhin liu ha thch ang ngy cng cn kit v hn na l tc ng xu n mi trng th nhin liu ethanol ngy cng c coi trng do tnh sch v an ton vi mi trng . Tuy nhin, khi y mnh sn xut ethanol bng cch s dng tinh bt nguy c din tch t nng nghip b thu hp dn n tinh trng thiu ht ngun lng thc. Ngun sinh khi lignocellulosic gi r, ti to c, sn lng di do c sn, v c kh nng chuyn i thnh glucose v mt vi loi ng khc c xem xt trong vi thp k qua, l mt con ng hp dn i vi sn xut ethanol. Qu trnh thy phn cellulose t nhin thnh glucose tri qua nhiu giai on v nh s kt hp ca 3 enzyme trong h thng cellulase, l -1,4-endoglucanase (EC 3.2.1.4), -1,4exoglucanase (EC 3.2.1.91) v cellobiase (EC 3.2.1.21). Chng Trichoderma reesei ZU-02 c bit n vi kh nng sn xut enzyme cellulase. Tuy nhin h enzyme ca chng vi sinh vt ny c khuyt im l hot tnh ca cc loi enzyme khng tng thch vi nhau dn n vic to thnh qu nhiu cellobio gy tc ch ngc nghim trng n cc phn ng ca -1,4-endoglucanase , -1,4-exoglucanase, nh hng n hiu sut chung ca qu trnh thy phn Trung Quc, lng ln cht thi nng nghip t li ng c sn sinh hng nm, l nguyn nhn chnh gy nhim mi trng do vic s dng thiu hiu qu. Trong bi ny, vic thy phn li bp bng enzmyme cellulase l s kt hp ca T. reesei ZU-02 v cellobiase t Aspergillus niger ZU-07 c nghin cu. Ngoi ra qu trnh ln men ca enzyme thy phn sn xut ethanol cng c th nghim bng nm men Saccharomyces cerevisiae 316. 2.3.1 Vt liu v phng php Vi sinh vt T. reesei ZU-02 (ngun gc t ATCC 56764) sn xut cellulase. A.niger ZU-07 (thu nhn t phng th nghim k thut ti to ngun ti nguyn, Purdue University) c s dng sn xut cellobiase. Chng S. cerevisiae 316 (c lu tr trong Phng th nghim K thut sinh ha, i hc Chit Giang) c s dng ln men sn xut ethanol. Vt liu lignocellulose v tin x l Li bp thu nhn ti a phng c phi kh. H2SO4 1% c cho vo vi t l rn:lng l 1:6. Hn hp c x l 108oC trong 3h sau em lc. D lng cellulose (sau tin x l) c ra vi nc, chnh pH 4.8 v sy kh, c cc thnh phn sau y: cellulose 59.4%, hemicellulose 6.5%, lignin 22.2%, v thnh phn khc 11,9%.

Sn xut cellulase Cellulase c sn xut bng ln men bn rn theo phng php ca Xia (Xia v Cen, 1999). Bo t ca T. reesei ZU-02 c cy vo mi trng ging v 30oC trong 48 gi iu kin hiu kh. Ln men rn, 10% ging lng c cho vo mi trng ln men (5cm) trong bnh erlen 1000 ml v nui trong t 28-30oC trong 7 ngy. Mi g koji kh cha 146 IU (FPU-Filter paper activity) v 12 IU (CBU-cellobiase activity). Sn xut cellobiase Cellobiase c sn xut theo phng php ca Shen (Shen v Xia, 2004). Huyn ph bo t ca A. niger ZU-07 c cy vo mi trng rn v nui 30oC trong 3 ngy. Hot tnh enzyme thu c t ln men rn cha 376 CBU/g\koji kh v khng pht hin FBU. 2.3.2 Enzyme thy phn d lng cellulose 2.3.2.1 Enzyme thy phn theo m M thy phn d lng cellulose li bp (sau tin x l) c thc hin trong bnh erlen 250 ml cha 100 ml hn hp nc v c cht rn. Cellulase t T. reesei ZU-02 v cellobiase t A. niger ZU-07 c s dng l enzyme thy phn. pH v nhit c iu chnh n 4.8 v 50oC. Cc bnh c trong mt my lc vi tc 160 rpm. Thi gian phn ng c thit lp 48h. 2.3.2.2 Enzyme thy phn fed-batch Qu trnh fed-batch c thc hin ti pH 4.8 v 50oC trong mt bnh phn ng 5 lt vi khi lng lm vic l 3 lt. Th nghim c bt u vi nng c cht t 100 g/l v enzyme cho vo c hot tnh 20 FPU/g c cht v 6.5 CBU/g c cht. D lng cellulose sau thm hai ln ti thi im 6 v 12 h c c mt nng c cht cui cng l 200 g/1, ng thi thm s lng nht nh cellulase v cellobiase (enzyme c 10 FPU/g c cht fed v 6.5 CBU/g c cht fed). Tng thi gian thy phn c thit lp trong 60h. 2.3.3 Sn xut ethanol Mu cy c chun b bng cch chuyn t bo S.cerevisiae 316 vo bnh nh mc

250 ml cha 50 ml mi trng nui cy (30 g/1 glucose, 5 g/1 peptone, 3 g/1 dch chit nm men), 30oC trong 24h. Cc t bo c thu li bng cch ly tm (4800 rpm, 5ph). T bo nm men c ra bng nc ct v s dng cy vo mi trng ln men. Dch thy phn cellulose sau qu trnh fed-batch, b sung vi 3 g/l dch chit nm men v 0.25 g/l (NH4)2HPO4, c s dng nh mt mi trng ln men. Qu trnh ln men

ethanol c thc hin 30oC trong iu kin k kh, vi 0.5 ml huyn ph t bo cy vo bnh100 ml vi khi lng lm vic l 50 ml. 2.3.4 Phng php phn tch Enzyme t koji c ly trch vi 50 phn th tch nc nhit phng trong 6 gi sau em lc. Dch lc c ly tm 4000 rpm trong 10 pht, v phn dch ni c s dng kho st hot tnh enzyme. FPU v CBU c xc nh theo tiu chun ca quy trnh IUPAC (Ghose, 1987). FPU c kho nghim bng cch mt hn hp phn ng gm: mt mnh giy lc Whatman no. 1 (16 cm) chm trong 1 ml m citrate 0.05M s dng 0.5 ml dung dch enzyme long nhit 50oC trong 30 pht. Mt n v FPU c nh ngha l lng enzyme m n to thnh 1mmol ng glucos/pht theo iu kin th nghim. CBU c kim nghim trong mt hn hp phn ng c cha 1 ml cellobiose 15 mM (trong m citrate 0.05M, pH 4.8) v 1 ml dch enzyme long thch hp c s dng, nhit ca phn ng l 50oC trong 30 pht. Mt n v CBU c nh ngha l lng enzyme m n to thnh 2 mmol glucose/mt pht t cellobiose. Lng ng kh c xc nh bng cch s dng phng php acid 3,5dinitrosalicylic (DNS) (Ghose, 1987). Glucose, xylose, cellobiose, arabinose, ethanol v glycerol c phn tch bng phng php HPLC (Syltech model 500 pump, USA) vi mt ct acid hu c (TRANSGENOMIC ICSep ICECOREGEL Column). Nc tinh khit c s dng nh l pha di ng vi tc dng chy 0,5 ml/pht. Nhit ct c c nh ti 60oC. Nc gii hp c khim tra bi mt my d ch s khc x (SpectraPhysics 6040 XR RI detector). Hiu sut thy phn enzyme c tnh nh sau: Hiu sut thy phn (%) = Lng ng kh 0.9 100 / lng polysaccharide trong c cht.t nht ba mu ng thi c s dng trong tt c cc php phn tch, v cc s liu c trnh by nh l trung bnh ca ba ln lp li. 2.3.5 Kt qu v bn lun 2.3.5.1 Thy phn tng m nh enzyme nh hng ca nng c cht Th nghim nh sau: c nh nng enzyme v thay i nng c cht. kt qu c hin th trn hnh 1.

Hnh 3.17 nh hng ca nng c cht n hot ng ca enzyme thy phn vi t l c nh ca T. reesei ZU-02 l 20FPUg- 1 cht nn; 1.64CBUg- 1 cht nn). Nhn xt: khi nng c cht tng hm lng ng tng nhng hiu sut thy phn li gim. iu ny c th gii thch l do s c ch ngc ca hm lng ng kh cao ln enzyme thy phn. T th da vo hm lng ng sinh ra v nng sut thy phn ta kt lun: nng c cht ti u l 100g/l. nh hng ca hm lng cellulase Chi ph nguyn liu chim mt phn ng k trong quy trnh sn xut bioethanol. V vy cellulose cn c phn hy cng trit cng tt. Th nghim thy phn c thc hin vi 100 gl-1 c cht v hm lng khc nhau ca T. reesei ZU 02 xenlulaza (hot tnh FPUg-1 cht nn) pH 4,8 v 500C, kt qu c th hin trong hnh. 2. Nng ng v hiu sut thy phn c xu hng bin i tng t, c ngha l, u tng mnh vi liu lng xenlulaza khc nhau t 10 n 20FPUg 1 b mt, v v c bn chng li 20 n 30FPUg 1 cht nn.

Hnh 3.18 nh hng ca hm lng Cellulase nh hng ca thi gian ln qu trnh thy phn cellulase bng enzyme Th nghim nh sau: tin hnh thy phn vi nng enzyme khng i (120FPUg-1 c cht hoc 1.64 CBUg-1 c cht) pH 4.8; nhit 500C trong 60h. hm lng cc cht sinh ra trong phn ng thy phn nh Glucose, xylose, arabinose v cellobiose c phn tch bng phng php HPLC c sau mi 12h. (bng 3.3) Bng 3.3 Qu trnh thy phn ca enzyme t T. reesei ZU-02 (20FPU/g c cht; 1.64 CBU/g c cht) Thi gian (h) 12 24 36 48 60 Nng ng trong qu trnh thy phn Cellobiose 3.7 7.4 8.6 11.7 12.2 Glucose 9.0 16.2 24.0 26.9 28.1 Xylose 1.7 3.9 5.4 6.7 6.9 Hm lng Nng sut Arobinose ng gim thy phn 0.5 0.7 0.7 0.9 0.9 16.9 30.8 41.2 49.4 51.1 23.1 42.1 56.3 67.5 69.8

Hnh 3.19 qu trnh thy phn fed-batch kt hp gia enzyme xenlul t T. reesei ZU-02 v cellobiase t A. niger ZU-07

T th ta thy: Hm lng ng kh sinh ra v hiu sut thy phn t tt nht thi im 48h. Trong qu trnh thy phn thy s hin din ca xylose v arabinose chng t c s tng hp enzyme xylanase T. reesei ZU-02. -1,4-endoglucanase v -1,4exoglucanase trong cellulase thy phn chui cellulose thnh cellubiose, cht ny sau c ct thnh glucose. Trong th nghim ny hm lng cellubiose tp trung rt cao cho thy hot tnh ca enzyme -glucosidase thp, khi hm lng cellubiose tp trung cao s c ch hot ng ca enzyme -1,4-endoglucanase v -1,4-exoglucanase v do nh hng n qu trnh thy phn. S kt hp ng thi cellulase t T. reesei ZU-02 v cellobiase t A. niger ZU-07 gim hin tng c ch ngc gy ra bi hm lng cao cellubiose, cellubiase sn xut bi A. niger ZU-07 hot ng v thy phn cellubio. Vi hot tnh cellulase l 20 FPUg-1 c cht. S gim nng ng v hiu sut thy phn tng ln cng vi s tng ln ca enzyme cellobiase n khi t nng 6.5CBU/g c cht. Khi tng hm lng enzyme hiu sut thy phn khng tng. Kt qu ca qu trnh thy phn vi s kt hp ng thi hai chng vi sinh vt c th hin trong bng 3.4. T s liu thu c ta nhn thy rng hm lng cellubiose thp trong ton b qu trnh, c th kt lun rng cellobiose hnh thnh trong qu trnh thy phn nhanh chng c thy phn thnh glucose nh vo tng cng enzyme cellobiase hot ng trong h thng thy phn. Qua , s c ch phn hi gy ra bi s tch t cellobiose c gim ng k, kt qu l nng ng kh v

nng sut thy phn cao hn. Lc 48 gi, sn lng thy phn c ci thin 83,9% v nng ng gim t 61,4 g/l. Bng 3.4 Kt qu ca s thy phn s dng hn hp 2 enzyme Thi gian (h) 12 24 36 48 60 Nng ng trong qu trnh thy phn Cellobiose 0.4 0.6 0.7 0.9 0.9 Glucose 18.8 34.1 43.7 50.1 51.2 Xylose 2.3 4.7 6.2 6.9 6.8 Hm lng Nng sut

Arobinose ng gim thy phn 0.7 0.8 0.8 1.0 1.0 24.3 42.3 53.7 61.4 62.2 33.2 57.8 73.4 83.9 84.9

2.3.5.2 Qu trnh thy phn Fed-batch vi xenlulaza v cellobiase Trong sn xut ethanol t nguyn liu lignocellulose, nng ethanol trong dung dch ln men nn cng cao cng gim thiu s hao ht do bc hi v hao ht trong qu trnh chng ct (Wingren et al, 2003), iu i hi nng ng ban u phi tng i cao. Tng nng c cht trong qu trnh thy phn l mt cch c c nng ng cao hn, nhng qu trnh thy phn thng xy ra khng trit l do vi khi lng ln c cht s o trn khng ng u v do s tip xc ca c cht v enzyme khng tt nh hng n hiu sut thy phn (Rudolf et al, 2005). Trong khi , s dng phng php ln men Fed- batch c th trnh c cc hin tng ny. Khi c cht dn cn kit v thay i (cc tnh cht vt l), nht ca hn hp phn ng c th c gi mc thp do qu trnh hon lu. Kt qu cho thy nng c cht tng ln 20 g/l trong qu trnh fed-batch, v nng ng gim t 116,3g/l vi nng sut thy phn l 79,5% sau 60 h phn ng (Hnh 3). Nng glucose trong dch thy phn t 95,3 g/l, thch hp cho qu trnh ln men ethanol bng S. cerevisiae. Hm lng Fed-batch l 15FPU/g. phng php ln men fed-batch gip rt ngn ng k thi gian ln men v do lm tng hiu sut ca qu trnh sn xut.

2.3.7 Ln men ethanol t dch cellulose thy phn. Dch thy phn cellulose giu glucose thu c t qu trnh trn c b sung chit xut nm men v (NH4) 2HPO4 sau c ln men ethanol bng S. cerevisiae . Kt qu ch ra rng S. cerevisiae 316 c th d dng ln men glucose sn xut ethanol nhng khng chuyn ha xylose do thiu h enzyme xylose reductase v xylitol dehydrogenase (Hnh 4). Trong thi gian 18 gi, 95.3 g/l glucose c ln men to ra 45,7 g/1 ethanol, tng ng vi 94% nng sut l thuyt (da trn nng sut l thuyt ca 0,51 ethanol g/g glucose).

Hnh 3.20 Ln men fed-batch dch thy phn cellulose bng S. cerevisiae 316.